Vectors for enhanced expression of VEGF for atrial disease treatment

ABSTRACT

The invention provides a vector which is capable of the expression of a vascular endothelial growth factor wherein the vector comprises a modified PCMV promoter. The invention further provides use of a vector which is capable of and expressing a vascular endothelial growth factor (VEGF) for the regulation of endothelial function, angiogenesis and arteriogenesis. The invention further comprises use of a vector which is capable of the expression of a vascular endothelial growth factor (VEGF) for the prophylactic treatment of arterial diseases and/or bone marrow diseases and/or neural diseases.

This application is a national stage entry under 35 U.S.C. §371 of PCTInternational Application No. PCT/NL02/00326, filed May 23, 2002, whichclaims priority of U.S. patent application Ser. No. 09/873,109, filedJun. 1, 2001 now abandoned, and of European Patent Application No.01201944.4, filed May 23, 2001, the contents of which are incorporatedherein by reference in their entirety.

The invention relates to the field of medicine. In particular it relatesto the treatment of disease more specifically arterial diseases and/orbone marrow diseases and/or neural diseases and/or inflammatorydisorders.

In mammalian vascular development mesoderm-derived cells differentiateinto endothelial cells that coalesce into blood vessels in a processcalled vasculogenesis. The formation of new blood vessels frompre-existing endothelium is termed angiogenesis. Several markers areconsistently associated with embryonic stem (ES) cell-derived precursorcells having vascular potential, including the vascular endothelialgrowth factor (VEGF) receptor flk-1, the cell adhesion receptor plateletendothelial cell adhesion molecule (PECAM), and the adhesion moleculeVE-cadherin.

VEGF-A functions in the development of embryonic structures duringtissue remodelling and for the growth of tumour-induced vasculature. Insolid tumors there is a constant requirement for vascular supply.Tumor-associated neovascularization is important for tumor cells toexpress their critical growth advantage. Experimental and clinicalevidence suggests that the process of metastasis, isangiogenesis-dependent. Angiogenesis is a crucial process for tumorgrowth, metastasis and inflammation. Various angiogenic growth factorsand cytokines induce neovascularization in tumors, namely members of thevascular endothelial growth factor (VEGF) and angiopoietin (Ang) genefamilies. Vascular endothelial growth factor (VEGF) is thought to be themost potent angiogenic factor in numerous malignant tumors and is aprognostic indicator for cancer patients. A strong correlation has beenfound between VEGF expression and increased tumor microvasculature,malignancy, and metastasis, for example in breast cancer. Vascularendothelial growth factor (VEGF) signaling is required for bothdifferentiation and proliferation of vascular endothelium. VEGF-Astimulates many actions of endothelial cells including proliferation,migration, and nitric oxide release via binding to and activation of thetwo primarily endothelial-specific receptor-tyrosine kinases KDR andFlt-1. KDR and Flt-1 stimulate multiple signal transduction pathways inendothelial cells. These molecules also have in vivo expression patternsthat are consistent with their being early markers of vascular lineage.VEGF signaling is critical for blood vessel formation duringdevelopment.

In mouse VEGF is a 45-kd homodimer produced at sites of vasculogenesisand angiogenesis, and alternative splicing results in 3 differentisoforms. The homodimer of VEGF-165 is the most active form and itsproperties include mitogenesis, chemotaxis, and permeability forendothelial cells. VEGF binds to 2 high-affinity receptors, flk-1(VEGFR-2) and flt-1 (VEGFR-1), that are expressed in endothelium.Recently, a third molecule that binds VEGF with high affinity wasidentified as neuropilin-1, a receptor that also signals in the nervoussystem through a different ligand. Available data suggest thatneuropilin-1 acts as a coreceptor with flk-1 in vascular tissues.Expression patterns of receptors and ligands suggest that VEGF may be ahighly specific mediator of blood vessel formation in vivo, and analysisof targeted mutations in the mouse supports this hypothesis. Both flk-1and flt-1 receptor mutations are recessive embryonic lethals at days 8.5to 9.5 of gestation. The flk-1 mutation severely impairs vasculogenesisand hematopoiesis, whereas the flk-1 mutation affects vascularorganization. Recent findings suggest that VEGF signaling is requiredfor the transition of flk-1+ and PECAM+ cells to vascular endothelialcells that express ICAM-2 and CD34.

Dominantly acting transforming oncogenes are generally considered tocontribute to tumor development and progression by their direct effectson tumor cell proliferation and differentiation. The growth of solidtumors beyond 1–2 mm in diameter requires the induction and maintenanceof a tumor blood vessel supply, which is attributed in large part to theproduction of angiogenesis promoting growth factors by tumor cells. Themechanisms which govern the expression of angiogenesis growth factors intumor cells are largely unknown, but dominantly acting oncogenes arethought to have a much greater impact than hitherto realised. An exampleof this is the induction of expression of vascular endothelial growthfactor/vascular permeability factor (VEGF/VPF) by mutant H- or K-rasoncogenes, as well as v-src and v-raf, in transformed fibroblasts orepithelial cells. Tumor-derived vascular endothelial growth factor(VEGF)/vascular permeability factor (VPF) plays an important role inneovascularization and the development of tumor stroma. BesidesVEGF/VPF, mutant ras genes are known to up-regulate the expression of avariety of other growth factors thought to have direct or indirectstimulating effects on angiogenesis, e.g. TGF-beta and TGF-alpha. Thiseffect may be mediated through the ras-raf-MAP kinase signaltransduction pathway, resulting in activation of transcription factorssuch as AP1, which can then bind to relevant sites in the promoterregions of genes encoding angiogenesis growth factors. In principle,similar events could take place after activation or over-expression ofmany other oncogenes, especially those which can mediate their functionthrough ras-dependent signal transduction pathways.

Excess blood vessel formation contribute to initiating and maintainingmany diseases such as chronic inflammatory disorders, tumor growth,restenosis, and atherosclerosis. In contrast insufficient blood vesselformation is responsible for tissue ischemia, as in coronary arterydisease. The treatment of vascular disease although greatly improvedover recent decades by drug medication, surgical and minimally-invasivetechniques, remains limited by vascular proliferative lesions and by ourinability to modulate the progression of native disease. An increasingnumber of patients with advanced coronary artery disease remainsymptomatic despite maximal interventional, surgical or medicaltreatment. Ideally, they would benefit most from additional arterialblood supply to ischemic areas of myocardium. The invention discloses anovel therapeutic strategy for the treatment of vascular disease usingthe angiogenic growth factor VEGF. Previous therapeutic strategies usingVEGF comprising proteins and/or plasmids were not able to produce VEGFpolypeptide in a mammalian cells in sufficient quantities to improveneovascularisation or regional myeardial blood flow.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Plasmid phVEGF165.MB sequence (SEQ ID NO:6)

The invention provides a vector which is capable of effecting theexpression of a vascular endothelial growth factor (VEGF or a functionalequivalent thereof) in the appropriate environment wherein said vectorcomprises a modified pCMV promoter. The definition “functionalequivalent” (homologue) means that a particular subject sequence variesfrom the reference sequence by one or more substitutions, deletions, oradditions resulting in a sequence that encodes the same activity as VEGFin kind, not necessarily in amount. A vector capable of the effectingthe expression of VEGF as used herein is a vector comprising a VEGEnucleic acid, that is at least capable of being expressed in theappropriate environment (i.e. a vector that is at least operative inmammalian systems). Preferably said vector comprises a plasmid.Preferably said modified pCMV promoter comprises the insertion of a 9 bpnucleic acid sequence ACGCGCGCT (SEQ ID NO:1), wherein A is adeoxyadenyl, G is deoxyguanyl, C is deoxycytosyl and T is thymidyl. pCMVpromoter refers to the human cytomegalovirus (CMV) promoter. It isunderstood that (single) base substitutions may be made in said promotersequence that will not significandy affect the function of the sequence.

The invention also provides a vector capable of effecting expression ofVEGF, which lacks CpG-rich regions. Prior art vectors often hadampicillin resistance sequences or other sequences promotingimmunogenicity such as CpG-islands. This may have contributed to thelack of efficacy in the prior art.

The invention provides a vector capable of effecting the expression of avascular endothelial growth factor (VEGF) wherein said vector comprisesan antibiotic resistance gene. Preferably said antibiotic resistancegene comprises kanamycin. Even more preferred said vector comprises a 5′HSV translation initiation signal and/or a β-globin splicing/adenylationsignal. Also considered are all viral and eukaryotic, preferablymammalian, translation initiation signals and splicing/adenylationsignals. Exogenous transcriptional elements and initation codons can beused and also can be of various origins, both natural and synthetic. Theinvention further provides a vector which is capable of effecting theexpression of a vascular endothelial growth factor (VEGF), wherein saidvector comprises a ColE1-like replicon and/or an F1 replication origin.ColE1-like replicons (e.g., pBR322) and other ColE1-like replicons(pMB1-, p15A, RSF1030-, and CloDF13-derived) in E. coli are consideredsuitable. F1 replication origin refers to phage F1 origin. All knownreplication origins previously defined and yet to be defined are deemedsuitable. The efficiency of expression may be enhanced by the inclusionof enhancers appropriate for and least operative in mammalian systems.

In a preferred embodiment the invention provides a vector capable ofeffecting the expression of a vascular endothelial growth factor (VEGF),wherein said vascular endothelial growth factor (VEGF) is derived from amammal. Preferably said mammal is a human. The invention furtherprovides a vector capable of effecting the expression of a vascularendothelial growth factor (VEGF), wherein said vector comprises avascular endothelial growth factor (VEGF) nucleic acid. Nucleic acid asused herein refers to an oligonucleotide, nucleotide or polynucleotide,and fragments or portions thereof, and to DNA or RNA of genomic orsynthetic origin which may be single- or double-stranded, and representsthe sense or antisense strand. It is understood that it is advantageousto increase the production of the endogenous vascular endothelial growthfactor (VEGF) in a mammalian system, to promote angiogenesis, forexample for the prophylactic treatment of arterial diseases and/or bonemarrow diseases and/or neural diseases and/or inflammatory disorders, inparticular tissue ischemia especially associated with diabetes. In apreferred embodiment said nucleic acid is operative in mammaliansystems.

In the case of mammalian expression vectors, the expression of a nucleicacid of the invention may be driven by a number of previously definedand yet to be defined promoters, including inducible, developmentallyregulated and tissue specific promoters. Promoters or enhancers derivedfrom the genomes of mammalian cells are considered suitable. Theinvention further provides the use of the individual promoter of thenucleic acid of the present invention for this purpose. In particularany promoters of nucleic acids particularly involved in the regulationof endothelial function, angiogenesis or arteriogenesis are deemedsuitable.

The invention provides a vector according to the invention wherein saidvector can replicate to a high copy number in a bacterial cell. Theinvention further provides a host cell which comprises a vectoraccording to the invention. The definition host cell as used hereinrefers to a cell in which an foreign process is executed bybio-interaction, irrespective of the cell belonging to a unicellular,multi-cellular, a differentiated organism or to an artificial cell orcell culture. Preferably said host cell is a mammalian cell, morepreferred a human cell. The invention provides a host cell whichcontains within its genome a recombinant nucleic acid according theinvention.

In a preferred embodiment the invention provides use of a vector whichis suitable and capable of effecting the expression of a vascularendothelial growth factor (VEGF) according to the invention for theregulation of endothelial function. Vascular endothelial growth factor(VEGF) is also a specific endothelial cell mitogen that stimulatesendothelial function and angiogenesis and plays a crucial role in tumorgrowth/angiogendsis. VEGF has a variety of effects on vascularendothelium, including the ability to promote endothelial cellviability, mitogenesis, chemotaxis, and vascular permeability. Vascularendothelial growth factor-A (VEGF-A) acts on endothelial cells andmonocytes, two cell types that participate in the angiogenic andarteriogenic process in vivo. Monocytes are important in arteriogenesisand that their ability to migrate may be critical to the arteriogenicresponse. VEGF-A stimulates monocyte migration in healthy individuals.

The invention further provides use of a vector which is suitable andcapable of effecting the expression of a vascular endothelial growthfactor (VEGF) according to the invention for the regulation ofangiogenesis. VEGF signaling is critical for blood vessel formationduring development. VEGF plays an important role in the development ofthe vascular system, wound healing, vascularization of tumors, and forangiogenesis in ischemic tissues including the heart. The inventionprovides for use of a vector according to the invention for promotingneovascularization of ischemic tissues, as a preventative treatment orearly stage treatment of cardiovascular diseases. VEGF-A can initiatethe process of vascularization by stimulating chemoattraction andproliferation of angioblasts and endothelial cells [i.e. VEGF-Aexpression can stimulate angiogenic remodeling]. VEGF is also a potentvasodilating agent. The invention provides for use of a vector accordingto the invention for promoting neovascularization of wounds. VEGFexpression is involved not only the formation of a vascular network butalso promotes tissue formation. VEGF is also required for the cyclicalblood vessel proliferation in the female reproductive tract.

The invention further comprises the use of a vector which is suitableand capable of effecting the expression of a vascular endothelial growthfactor (VEGF) according to the invention for the regulation ofarteriogenesis. In a preferred embodiment the invention provides the useof a vector according to the invention for the regulation of coronaryand peripheral artery angiogenesis. VEGF-A plays a role as an endogenousactivator of coronary collateral formation in the human heart. In apreferred embodiment said vector capable of effecting the expression ofVEGF according the invention can be used in the prophylactic treatmentof myocardial tissue (diseases), and also in the reconstructing processof infarcted myocardial tissue.

The invention further provides use of a vector according to theinvention to induce the release of hematopoietic growth factors by bonemarrow endothelial cells. VEGF is also required for longitudinal bonegrowth and endochondral bone formation. VEGF can induce the release ofhematopoietic growth factors (GM-CSF) by bone marrow endothelial cells.In a preferred embodiment said vector capable of effecting theexpression of VEGF according to the invention can be used to induce therelease of hematopoietic growth factors, for example GM-CSF, toaccelerate hematologic recovery after bone marrow grafting.Hematopoietic growth factors can mobilize peripheral blood stem cellsfrom the bone marrow to therapeutically intervene in acceleratinghematologic recovery. In a preferred embodiment said vector capable ofeffecting the expression of VEGF according to the invention may be usedfor the prophylactic treatment of haematological and oncologicaldiseases. Preferably said vector capable of effecting the expression ofVEGF according to the invention may be used to release hematopoieticgrowth factors in tissues after myeloablative chemotherapy andhematopoietic stem cell grafting. This can reduce the mortality relatedto autologous and allogeneic graft failure (i.e. bone marrow grafting).The invention further provides a vector capable of effecting theexpression of VEGF according to the invention may be used to releasehematopoietic growth factors which may be used to increase the qualityof cytapheresis peripheral stem cell harvesting.

The invention further provides use of a vector capable of effecting theexpression of VEGF according to the invention to induce transendothelialprogenitor cell migration. Preferably to induce stromal cell-derivedfactor-1 (SDF-1) driven transendothelial progenitor cell migration. Inthe presence of VEGF in-vitro stromal cell-derived factor-1 (SDF-1) candrive and thus increase transendothelial progenitor cell migration. Thismay be due to pore formation (increased endothelial fenestration).Transendothelial migration of progenitors in vitro is substantiallyenhanced by the chemokine stromal-cell-derived factor-1 (SDF-1), whichis produced by bone marrow stromal cells. More primitive progenitorsalso respond to this chemokine. Transendothelial progenitor cellmigration is regulated by adhesion molecules, paracrine cytokines, andchemokines. Mobilizing hematopoietic growth factors stimulateproliferation of hematopoietic cells, which may indirectly result inchanges of the local cytokine and chemokine milieu, adhesion moleculeexpression, and eventually the mobilization of hematopoietic progenitorcells.

The invention provides use of a vector capable of effecting theexpression of VEGF according to the invention to increase endothelialfenestration. In tissues outside the brain, vascular endothelial growthfactor-A (VEGF) causes vascular hyper-permeability by opening ofinter-endothelial junctions and induction of fenestrations andvesiculo-vacuolar organelles (VVOs).

The invention provides use of a vector capable of effecting theexpression of VEGF according to the invention to modulate neuroblastomacell growth. VEGF may affect neuroblastoma cell growth directly andcould be an autocrine growth factor. In a preferred embodiment saidvector capable of effecting the expression of VEGF according theinvention can be used in the prophylactic treatment of neural diseases,and in the reconstruction process after neural injury.

Critical Limb Ischemia and Diabetes Mellitus

Critical limb ischemia is one of the most burdensome problems indiabetes treatment, and provides a major challenge for VEGF genetransfer:

-   -   Patients with diabetes mellitus have a 20-fold risk of        developing clinical peripheral arterial disease, a 17-fold        increased risk of developing foot gangrene, and a 15-fold        increased risk of amputation. In the Netherlands in 1992, 1810        amputations were performed. Actually 50% of lower leg        amputations in non-trauma patients is performed in diabetes        mellitus. Patients with diabetes mellitus have after unilateral        amputation a 40–55% risk of amputation of the contralateral limb        in the next 5 years. While the mean admission duration for        clinical cases with a primary diagnosis of peripheral arterial        disease is 12.2 days, this increases to 30 days in diabetes        mellitus. Thus, in diabetes mellitus the prevalence of CLI is        not only increased, but its course is markedly more serious.    -   The localization of macrovascular lesions (stenosis/occlusions)        in diabetes mellitus shows a predilection for distal vessels.        Especially involvement of vessels below the knee is prominent.        On top of this, microvascular involvement is specific for        diabetes mellitus. The localization of these lesions makes them        less accessible to conventional forms of revascularisation        (surgery, PTA, stent).    -   The current range of treatment options in CLI in diabetic foot        disease is quite small. If possible revascularisation procedures        are performed, and in case of superimposed infections aggressive        antibiotic treatment is given. However, medical treatment is        limited to prostacyclin analogues and attempts to improve tissue        oxygenation with rheological means (e.g. Isodex), both with        limited success. Initial results from a clinical study show less        amputations and better limb survival of as a result of the VEGF        treatment for the mentioned disease. Thus gene transfer of a        VEGF vector according to the invention is useful for the        treatment of tissue ischemia.

The invention further provides use of a vector capable of effecting theexpression of VEGF according to the invention in the preparation of acomposition. Suitable base for compositions are known in the art. Theinvention further provides a composition comprising a vector capable ofeffecting the expression of VEGF according to the invention. Preferablysaid composition is in a form that can be administered to a mammal. Apreferred embodiment is that said composition is suitable for humanexternal application, for example wound healing.

The invention also provides a composition comprising a vector capable ofeffecting the expression of VEGF according to the invention which is apharmaceutical. Suitable pharmaceutical compositions are known and theymay be in dosage forms such as tablets, pills, powders, suspensions,capsules, suppositories, injection preparations, ointments, eye dropsetc.

In a preferred embodiment the invention provides use of a compositionaccording to the invention and/or a vector according to the inventionfor the treatment of arterial diseases and/or bone marrow diseasesand/or neural diseases and/or inflammatory disorders, in particulartissue ischemia especially associated with diabetes. Modes ofadministration can readily be determined by conventional protocols. Apreferable mode of administration is by syringe injection to a localizedtissue.

The invention further comprises a method of treatment of arterialdiseases and/or bone marrow diseases and/or neural diseases and/orinflammatory disorders and/or wounds comprising administering a vectoraccording to the invention and/or a composition according to theinvention with a carrier to a suitable recipient. Preferably saidcarrier is a pharmaceutically acceptable carrier (e.g. drug carriersystem) or inert carrier.

EXAMPLES Example 1 Construction of a Highly Efficient VEGF ExpressionPlasmid (phVEGF165.MB) Without an Ampicilline Resistant Gene

Plasmid phVEGF165.MB (FIG. 1) was constructed from phVEGF165.SR, whichwas originally constructed by cloning a VEGF cDNA, obtained by RT-PCR ofhuman vascular smooth muscle cells, into a eukaryotic expression vector(see Severne et al., 1988 and Isner et al., 1996 for details). Theplasmid phVEGF165.SR (kindly provided by Isner), was re-sequenced.Besides a few mutations (including in the A-hdI site), an almostcomplete β-lactamase gene was found. The 3′ part of the ampicillinresistance gene (751 bp) was deleted out of phVEGF165.SR.

Deleting of the 3′ part of β-lactamase gene (751 bp)/Creating AhdI site:

The PCR-forward primer: 5′-ATCGACATCCAGTCAAAGCCACGTTGTGTCTCA-3′ (SEQ IDNO:2) (first 2 nucleotides are miscellaneous, followed by an AhdI site),and the PCR reverse primer: 5′-ATAGCATGCGAGTTTCGCCCCGAAGA-3′ (SEQ IDNO:3) were used to amplify kanamycin.

For ColE1-origin the forward primer:5′-GCGGACTAGTGCTGTCCCTCTTCTCTTATGA-3′ (SEQ ID NO:4) and the backward orreversed primer: 5′GCCGACGTGCAGTCTAGGTGAAGATCCTTTT-3′ (SEQ ID NO:5)(increased with AhdI) were used.

Both PCR products were ligated to each other, using AhdI, in anpCR-BluntII-TOPO (Invitrogen). And after sequencing, the kanamycin/origene was digested with ClaI and BlnI, and ligated into the ClaI/BlnIsites of phVEGF165.SR. This resulted in a plasmid without ampicillin andwith a new AhdI restriction site: phVEGF165.MB. The promotor region waschanged during growth in the E. Coli DH5-alpha and included in thispromoter was a 9 bp nucleic acid sequence ACGCGCGCT (SEQ ID NO:1)insertion. Synthesis of VEGF mRNA is driven by the modifiedcytomegalovirus (CMV) promoter. Downstream of the VEGF coding region isa rabbit β-globin sequence for splicing and polyadenylation. A 5′untranslated thymidine kinase sequence from herpes simplex virus (HSV)is used for translation initiation. The phVEGF165.MB plasmid harboursthe kanamycin gene for selection during propagation in E. Coli.

Positions in the plasmid:  14–619 CMV promoter/enhancer CMV 620–6825′HSV translation initiation signal 5′HSV  682–1258 VEGF coding sequenceVEGF 1259–2180 β-globin splicing/polyadenylation signal pA 2457–3370ColE1-like replicon ori 3388–4623 kanamycin gene kana 4647–4724 5′ partof β-lactamase gene 5238–5690 f1 origin f1 ori 5854–5875 T7 promoter T7

Example 2 Detection of the Ability of the phVEGF165MB Plasmid toTransform COS7 Cells to Produce VEGF Protein

70% confluent COS7 cells were transfected with VEGF plasmid Fugene.After 3 and 4 days the VEGF concentration in the supernatant wasdetected with a VEGF R&D kit (ELISA). The VEGF production (n=3) on day 3and 4 of the plasmids without (phVEGF165.SR) and with (phVEGF165.MB) themodified cytomegalovirus (CMV) promoter were respectively 1.3/1.5 and1.4/1.6 (pg/ml per 10E6 COS cells).

Phase I clinical studies have established that intramuscular genetransfer may be utilized to successfully accomplish therapeuticangiogenesis.

Gene transfer was performed in ten limbs of nine patients withnon-healing ischemic ulcers (n=7) and/or rest pain (n=10) due toperipheral arterial disease. A total amount of 4000 μg naked plasmid DNAencoding the secreted 165-amino acid isoform of human VEGF (phVEGF₁₆₅)was injected into ischemic muscles of the affected limb. The averagefollow-up was 6±3 (range 2 to 11) months. Local intramuscular genetransfer induced no or mild local discomfort up to 72 hours after theinjection. Serial CPK measurements remained in the normal range andthere were no signs of systemic or local inflammatory reactions. Todate, no aggravated deterioration in eyesight due to diabeticretinopathy or growth of latent neoplasm has been observed in anypatient treated with phVEGF₁₆₅ gene tranfer. The only complicationobserved in the trial, was limited to transient lower extremity edema,consistent with VEGF-enhancement of vascular permeability.

Transgene expression. Blood levels of VEGF transiently peaked one tothree weeks post gene transfer in seven patients amenable for weeklyassays. In two patients baseline and/or more than two follow-up bloodsamples were not achievable. Clinical evidence of VEGF (vascularpermeability factor) overexpression was evident by the observation ofperipheral edema development (+1 to +4 by gross inspection) in those sixpatients with ischemic ulcers. In four patients, the edema was limitedto the treated limb, while in two patients the contralateral limb wasaffected as well, albeit less severely. Edema corresponded temporally tothe rise in serum VEGF levels.

Noninvasive arterial testing. The absolute systolic ankle or toepressure increased in nine limbs post gene transfer and was unchanged inone limb at the time of the most recent follow-up (p=0.008). The ABIand/or TBI increased from 0.33±0.04 (0.22 to 0.57, p=0.028, [n=10]) atfour weeks; to 0.45±0.04 (0.27 to 0.59, p=0.016, [n=10]) at eight weeks;and to 0.48±0.03 (0.27 to 0.67, p=0.017, [n=8]) at 12 weeks. Improvementin the pressure index was sustained, but did not further risesignificantly after the second gene transfer.

Exercise performance improved in all five patients with rest pain orminor ischemic ulcers, who underwent a graded treadmill exercise. Allpatients experienced a significant increase in pain-free walking time(2.5±1.1 min pre gene therapy vs 3.8±1.5 min at an average of 13 weekspost gene therapy, p=0.043) and absolute, claudication-limited walkingtime (4.2±2.1 min vs 6.7±2.9 min, p=0.018). Two patients reached thetarget endpoint of ten minutes of exercise.

Angiography. Digital subtraction angiography showed newly visiblecollateral vessels at the knee, calf and ankle levels in six of tenischemic limbs treated. The luminal diameter of the newly visiblevessels ranged from 200 μm to >800 μm, although most were closer to 200μm and these frequently appeared as a “blush” of innumerablecollaterals. Collaterals did not regress in follow-up angiograms.Magnetic resonance angiography showed qualitative evidence of improveddistal flow with enhancement of signal intensity as well as an increasein the number of newly visible collaterals in eight limbs.

Change in limb status and ischemic rest pain. Therapeutic benefit wasdemonstration by regression of rest pain and/or improved limb integrity.The frequency of ischemic rest pain expressed as afflicted nights perweek decreased significantly (5.9±2.1 at baseline vs 1.5±2.8 at eightweek follow-up, p=0.043), with a slight reduction of analgesicmedication (on average from 1.8 to 1.5 analgetics/24 h period). Based oncriteria proposed by Rutherford, limb status improved in nine of tenextremities treated. Moderate improvement, including both an upwardshift in the clinical category (at least one clinical category inpatients with rest pain and at least two categories to reach the levelof claudication in patients with tissue loss) and an increase in theABI>0.1 was documented in five cases. In one patient an ischemic ulcerresolved sufficiently to permit placement of a split-thickness skingrafting, leading to absolute limb salvage. In two patients, in whom amajor amputation would have been inevitable, retention of a functionalfoot by a minor (toe) amputation was reached. Minimal improvement,including an upward shift in clinical category or improvement of theABI>0.1 was present in another three cases. However, in two patientswith and extensive forefoot necrosis and osteomyelitis a below-kneeamputation was required despite significant hemodynamic and angiographicimprovement. There was one patient with progressive toe gangrene, whoremained unchanged from his hemodynamic and angiographic findings. Thepatient underwent a below-knee amputation eight weeks after genetherapy.

Immunohistochemistry and molecular analysis. Tissue specimens derivedfrom one amputee ten weeks after gene therapy showed foci ofproliferating endothelial cells. This finding was particularly strikingwith the fact in mind, that endothelial cell proliferation is nearlyabsent in normal arteries, is consistent with an estimated endotheliascell turnover time of “thousands of days” is quiescent microvasculature.PCR performed on these samples indicated persistence and widespreaddistribution of DNA fragments unique to phvEGF₁₆₅. Noteworthyamplification of DNA fragments was shown in muscle and skin samplesderived from the site of injection as well as in several muscle samplesremote from the site of injection. Southern blot analysis confirmedpersistence of intact plasmid DNA in muscle specimen derived from twoamputees eight and ten weeks after gene therapy.

Example 3 A Further Study was Performed to Determine the Effect ofTreating Diabetic Patients with critical Limb Ischemia with VEGF

Ischemic muscle represents a promising target for gene therapy withnaked plasmid DNA. Intramuscular (IM) transfection of genes encodingangiogenic cytokines, particularly those which are naturally secreted byintact cells, may constitute an alternative treatment strategy forpatients with extensive tissue ischemia, in whom contemporary therapies(pharmacologic interventions, angioplasty, bypass surgery) havepreviously failed or are not feasible. This strategy is designed topromote the development of supplemental collateral blood vessels thatwill constitute endogenous bypass conduits around occluded nativearteries, a strategy termed “therapeutic angiogenesis”.

Pre-clinical animal studies have indeed indicatedd that IM gene transfermay be utilized to successfully accomplish therapeutic angiogenesis.More recently, phase I clinical studies have indicated that IM genetransfer may be utilized to safely and successfully accomplishtherapeutic angiogenesis in patients with critical limb ischemia.

At this moment there is no standard pharmacotherapeutic treatment.Antithrombotic and vasoactive drugs are of little value in themanagement of CLI. Only prostanoids have shown some efficacy, but thereis no evidence that in the long-term the limb is saved.

The protocol outlined has been designed as a double blind placebocontrolled phase III study of direct intramuscular gene transfer ofphVEGF₁₆₅ in patients with critical limb ischemia (CLI).

The objectives are to determine the clinical response and physiologicextent of collateral artery development in patients receivingintramuscular phVEGF₁₆₅ gene transfer and the safety of this treatmenton diabetic retinopathy and nephropathy compared with placebo. Theprimary endpoints are limb survival 100 days after the first IMinjection with VEGF and an increase of ABI of 15%. A total of 60 adultmen and women will participate in this study. Subjects will be eligibleif they have critical limb ischemia, diabetes and not to be optimalcandidates for surgical or percutaneous revascularization. The clinicalresponse of subjects treated in this fashion will be evaluated by serialstudies performed before and after treatment, non invasive perfusiontechniques, capillary microscopy and PET.

Protocol

I. Therapeutic Angiogenesis is a Novel Strategy for the Treatment ofIsehemia.

The therapeutic implications of angiogenic growth factors wereidentified by the pioneering work of Folkman and colleagues over twodecades ago (1). Their work documented the extent to which tumordevelopment was dependent upon neovascularization and suggested thatthis relationship might involve angiogenic growth factors which werespecific for neoplasms. Beginning a little over a decade ago (2), aseries of polypeptide growth factors were purified, and demonstrated tobe responsible for natural as well as pathologic angiogenesis.

Subsequent investigations have established the feasibility of usingrecombinant formulations of such angiogenic growth factors to expediteand/or augment collateral artery development in animal models ofmyocardial and hindlimb ischemia. This novel strategy for the treatmentof vascular insufficiency has been termed “therapeutic angiogenesis”(3). The angiogenic growth factors first employed for this purposecomprised members of the FGF family. Baffour et al administered bFGF indaily intramuscular doses of 1 or 3 μg to rabbits with acute hindlimbischemia; at the completion of 14 days of treatment, angiography andnecropsy measurement of capillary density showed evidence of augmentedcollateral vessels in the lower limb, compared to controls (4). Pu et alused an acidic fibroblast growth factor (aFGP) to treat rabbits in whichthe acute effects of surgically-induced hindlimb ischemia were allowedto subside for 10 days before beginning a 10-day course of daily 4 mg IMinjections; at the completion of 30 days follow-up, both angiographicand hemodynamic evidence of collateral development was superior toischemic controls treated with IM saline (5). Yanagisawa-Miwa et allikewise demonstrated the feasibility of bFGF for salvage of infarctedmyocardium, but in this case growth factor was administeredintra-arterially at the time of coronary occlusion, followed 6 hrs laterby a second intra-arterial bolus (6).

Isner used the same animal model developed by Pu et al (5) toinvestigate the therapeutic potential of a 45 kDa dimeric glycoprotein,vascular endothelial growth factor (VEGF), isolated initially as aheparin-binding factor secreted from bovine pituitary folliculo-stellatecells (7). VEGF was also purified independently as a tumor-secretedfactor that induced vascular permeability by the Miles assay (8, 9), andthus its alternate designation, vascular permeability factor (VPF). Twofeatures distinguish VEGF from other heparin-binding, angiogenic growthfactors. First, the NH₂ terminus of VEGF is preceded by a typical signalsequence; therefore, unlike bFGF, VEGF can be secreted by intact cells(10). Second, its high-affinity binding sites, shown to include thetyrosine kinase receptors Flt-1 (11) and Flk-1/KDR (12, 13) are presenton endothelial cells, but not other cell types; consequently, themitogenic effects of VEGF—in contrast to acidic and basic FGF, both ofwhich are known to be mitogenic for smooth muscle cells (14, 15) andfibroblasts as well as endothelial cells—are limited to endothelialcells (7, 16). (Interaction of VEGF with lower affinity binding siteshas been shown to induce mononuclear phagocyte chemotaxis) (17, 18).Evidence that VEGF stimulates angiogenesis in vivo had been developed inexperiments performed on rat and rabbit cornea (19, 20), thechorioallantoic membrane (7), and the rabbit bone graft model (20). Thehypothesis was that the angiogenic potential of VEGF was sufficient toconstitute a therapeutic effect (21). This hypothesis was confirmedafter the administration of soluble 165-amino acid isoform of VEGF(VEGF₁₆₅) as a single intra-arterial bolus to the internal iliac arteryof rabbits in which the ipsilateral femoral artery was excised to inducesevere, unilateral hindlimb ischemia. Doses of 500–1.000 μg of VEGFproduced statistically significant augmentation of angiographicallyvisible collateral vessels, and histologically identifiable capillaries;consequent amelioration of the hemodynamic deficit in the ischemic limbwas significantly greater in animals receiving VEGF than in non-treatedcontrols (calf blood pressure ratio=0.75±0.14 vs 0.48±0.19, p<0.05).Serial (baseline, as well as 10 and 30 days post-VEGF) angiogramsdisclosed progressive linear extension of the collateral artery oforigin (stem artery) to the distal point of parent-vessel (reentryartery) reconstitution in 7 of 9 VEGF-treated animals. Similar resultswere achieved in a separate series of experiments in which VEGF wasadministered by an intramuscular route daily for 10 days (22). Thesefindings thus established proof of principle for the concept that theangiogene activity of VEGF is sufficiently potent to achieve therapeuticbenefit.

While each of these studies documented an increase in the number ofangiographically visible collaterals, and increased capillary density inthe muscles studied at necropsy, evidence regarding the physiologicalconsequences of such anatomical improvement was limited to bloodpressure measurements recorded in the ischemic versus the normal limb.Accordingly, a series of studies in the ischemic hindlimb model in whichan intra-arterial Doppler wire (23), sufficiently diminutive (0.018 in.)to measure phasic blood flow velocity in the rabbit's internal iliacartery, was used to investigate resting and maximum flow followingtherapeutic angiogenesis with a single, intra-arterial bolus of VEGF₁₆₅.By 30 days post-VEGF₁₆₅, flow at rest, as well as maximum flow velocityand maximum blood flow provoked by 2 mg papaverine were allsignificantly higher in the VEGF-treated group (24).

One of the distinguishing features of VEGF mentioned above—the fact thatthe VEGF gene encodes a secretory signal sequence—might be exploited aspart of a strategy designed to accomplish therapeutic angiogenesis byarterial gene transfer. Previously was observed that site-specifictransfection of rabbit ear arteries with the plasmid pXGH5 encoding thegene for human growth hormone—a secreted protein—yields local levels ofhuman growth hormone equivalent to what has been considered to be in aphysiologic range, despite the fact that immunohistochemical examinationof the transfected tissue disclosed evidence of successful transfectionin <1% of cells in the transfected arterial segment (25). Thus, geneproducts which are secreted may have profound biological effects, evenwhen the number of transduced cells remains low. In contrast, for genessuch as bFGF which do not encode a secretory signal sequence,transfection of a much larger cell population might be required for thatintracellular gene product to express its biological effects.

Therefore, 400 μg of phVEGF₁₆₅ was applied, encoding the 165-amino acidisoform of VEGF, to the hydrogel layer coating the outside of anangioplasty balloon (26) and delivered the balloon catheterpercutaneously to the iliac artery of rabbits in which the femoralartery had been excised to cause hindlimb ischemia. Site-specifictransfection of phVEGF₁₆₅ was confirmed by analysis of the transfectedinternal iliac arteries using reverse transcriptase-polymerase chainreaction (RT-PCR) and then sequencing the RT-PCR product. Augmenteddevelopment of collateral vessels was documented by serial angiograms invivo, and increased capillary density at necropsy. Consequentamelioration of the hemodynamic deficit in the ischemic limb wasdocumented by improvement in the calf blood pressure ratio(ischemic/normal limb) to 0.70±0.08 in the VEGF-transfected group vs0.50±0.18 in controls (p<0.05). Algiographic and histologic evidence ofangiogenesis were subsequently demonstrated following intra-arterialgene transfer of phVEGF₁₆₅ in a human patient (27). These findingsestablished that site-specific gene transfer can be used to achievephysiologically meaningful therapeutic modulation of vascular disorders,including therapeutic angiogenesis. Of note, no study have disclosed anyevidence of immunologic toxicity.

II. Critical Limb Ischemia and Diabetes Mellitus

Critical limb ischemia is one of the most burdensome problems indiabetes treatment, and provides a major challenge for VEGF genetransfer:

-   -   Patients with diabetes mellitus have a 20-fold risk of        developing clinical peripheral arterial disease, a 17-fold        increased risk of developing foot gangrene, and a 15-fold        increased risk of amputation. In the Netherlands in 1992 1810        amputations were performed. Actually 50% of lower leg        amputations in non-trauma patients is performed in diabetes        mellitus. Patients with diabetes mellitus have after unilateral        amputation a 40–55% risk of amputation of the contialateral limb        in the next 5 years. While the mean admission duration for        clinical cases with a primary diagnosis of peripheral arterial        disease is 12.2 days, this increases to 30 days in diabetes        mellitus. Thus, in diabetes mellitus the prevalence of CLI is        not only increased, but its course is markedly more serious.    -   The localization of macrovascular lesions (stenosis/occlusions)        in diabetes mellitus shows a predilection for distal vessels.        Especially involvement of vessels below the knee is prominent.        On top of this, microvascular involvement is specific for        diabetes mellitus. The localization of these lesions makes them        less accessible to conventional forms of revascularisation        (surgery, PTA, stent).    -   The current range of treatment options in CLI in diabetic foot        disease is quite small. If possible revascularisation procedures        are performed, and in case of superimposed infections aggressive        antibiotic treatment is given. However, medical treatment is        limited to prostacyclin analogues and attempts to improve tissue        oxygenation with rheological means (e.g. Isodex), both with        limited success.    -   On the other hand, other diabetic complications may increase the        risk of side effects of VEGF therapy in diabetes patients.        Especially proliferative retinopathy, but also less outspoken        forms of retinopathy and microalbuminuria might exacerbate in        case of systemic effects of VEGF. Even the nature of the local        abnormalities in CLI in diabetes might compromise beneficial        effects of VEGF: in diabetes mellitus total skin flow is not        necessarily reduced, but the contribution of nututive capillary        versus shunt skin flow may be lowered. It is at this stage not        known what effect intramuscular administration of VEGF may have        on this distribution of skin flow, and, clinically, on ulcer        healing.

In conclusion, CLI in patients with diabetes there is no standardtreatment, VEGF may be increase both the chance of beneficial andunwanted effects of VEGF in patients with diabetes mellitus (41,42,43).

III. Gene Transfer of cDNA Encoding for Secreted Protein.

In experiments which have relied exclusively on the use of non-secretedgene products, examination by histochemical staining, in situhybridization, and/or polymerase chain reaction has suggested that thetransfection efficiency of direct gene transfer to vascular smoothmuscle cells within the arterial wall was considerably less than 1% andmight therefore preclude a meaningful biological response. In contrast,genes encoding for a secreted protein may overcome the handicap ofinefficient transfection by a paracrine effect, secreting adequateprotein to achieve local levels that may be physiologically meaningful.Nabel et al (28) demonstrated that despite similarly low efficiencies,cell surface protein expression resulting from percutaneous transfectionof vascular smooth muscle cells with the histocompatibility gene HLA-B7may be adequate to induce a biological response, namely, focalvasculitis. Necropsy evidence of a pathobiological response followingarterial gene transfer was reported by the same group in the case oftransgenes encoding for the secreted proteins PDGF-B (29) and FGF-1(30); in the former study, only 0.1 to 1% of cells in the artery segmentwere estimated to contain plasmid DNA by PCR approximation. To morespecifically determine the relation between a secreted gene product andtransfection efficiency after in vivo arterial gene transfer, in vitro(31) and in vivo (25) models to serially monitor expression of a geneencoding for a secreted protein. In vivo analyses were performed usingthe central artery of the rabbit ear. Liposome-mediated transfection ofplasmid DNA containing the gene for human growth hormone (hGH) wassuccessfully performed in 18 of 23 arteries. Serum hGH levels measured 5days after transfection ranged from 0.1 to 3.8 ng/mL (mean, 0.97 ng/mL);in contrast, serum drawn from the control arteries demonstrated noevidence of hGH production. Serial measurement of hGH from transfectedarteries demonstrated maximum hGH secretion 5 days after transfectionand no detectable hormone after 20 days. Despite these levels ofsecreted gene product documented in vivo, immunohistochemical stainingof sections taken from the rabbit ear artery at necropsy disclosedevidence of successful transfection in <0.1% of cells in the transfectedsegment. Thus, low-efficiency transfection with a gene encoding for asecreted protein may achieve therapeutic effects not realized bytransfection with genes encoding for proteins which remainintracellular.

In conclusion—in combination with the fact that ischemic skeletal muscleitself serves to augment transfection efficiency (32, 33)—similarlyaccounts for the bioactivity that is described above of gene transfer ofnaked DNA by direct injection into skeletal muscle (vide infra).

IV. Pre-clinical Animal Studies.

Ten days after ischemia was induced in one hindlimb of New Zealand Whiterabbits, 500 μg of phVEGF₁₆₅, or the reporter gene LacZ, were injectedIM into the ischemic hindlimb muscles. Site-specific transgeneexpression was documented by mRNA and immunohistochemistry. At 30-dayfollow-up, angiographically recognizable collateral vessels andhistologically identifiable capillaries were increased inVEGF-transfectants compared to controls. This augmented vascularityimproved perfusion to the ischemic limb, as documented by a superiorcalf blood pressure ratio for phVEGF₁₆₅ (0.84±0.09) vs controls(0.67±0.06, p<0.1); by improved blood flow in the ischemic limb(measured using an intra-arterial Doppler wire) at rest(phVEGF₁₆₅=52.5±12.6, control=38.4±4.3, p<0.05); and by increaseddistribution of labeled microspheres to the adductor muscle(phVEGF₁₆₅=4.3±0.5, control=2.9±0.6 ml/min/100 g tissue, p<0.05), aswell as the gastrocnemius muscle (phVEGF₁₆₅=3.9±0.8, control=2.8±0.9ml/min/100 g tissue, p<0.05) of the ischemic limb. A more detaileddescription of this study has been published previously (32).

Ischemic muscle thus represents a promising target for gene therapy withnaked plasmid DNA. IM transfection of genes encoding angiogeniccytokines, particularly those which are naturally secreted by intactcells, may constitute an alternative treatment strategy for patientswith extensive tissue ischemia, in whom contemporary revascularization(anti-anginal medications, angioplasty, bypass surgery) have previouslyfailed or are not feasible.

V. Phase I Clinical Studies.

Gene transfer was performed in ten limbs of nine patients withnon-healing ischemic ulcers (n=7) and/or rest pain (n=10) due toperipheral arterial disease. A total amount of 4000 μg naked plasmid DNAencoding the secreted 165-animo acid isoform of human VEGF (phVEGF₁₆₅)was injected into ischemic muscles of the affected limb. The averagefollow-up was 6±3 (range 2 to 11) months. Local intramuscular genetransfer induced no or mild local discomfort up to 72 hours after theinjection. Serial CPK measurements remained in the normal range andthere were no signs of systemic or local inflammatory reactions. Todate, no aggravated deterioration in eyesight due to diabeticretinopathy or growth of latent neoplasm has been observed in anypatient treated with phVEGF₁₆₅ gene transfer. The only complicationobserved in the trial, was limited to transient lower extremity edema,consistent with VEGF-enhancement of vascular permeability (Baumgartneret al, Circ 1998; 97: 114–1123).

Transgene expression. Blood levels of VEGF transiently peaked one tothree weeks post gene transfer in seven patients amenable for weeklyassays. In two patients baseline and/or more than two follow-up bloodsamples were not achievable. Clinical evidence of VEGF (vascularpermeability factor) overexpression was evident by the observation ofperipheral edema development (+1 to +4 by gross inspection) in those sixpatients with ischemic ulcers. In four patients, the edema was limitedto the treated limb, while in two patients the contralateral limb wasaffected as well, albeit less severely. Edema corresponded temporally tothe rise in serum VEGF levels.

Noninvasive arterial testing. The absolute systolic ankle or toepressure increased in nine limbs post gene transfer and was unchanged inone limb at the time of the most recent follow-up (p=0.008). The ABIand/or TBI increased from 0.33±0.04 (0.22 to 0.57, p=0.028, [n=10]) atfour weeks; to 0.45±0.04 (0.27 to 0.59, p=0.016, [n=10]) at eight weeks;and to 0.48±0.03 (0.27 to 0.67, p=0.017, [n=8]) at 12 weeks. Improvementin the pressure index was sustained, but did not further risesignificantly after the second gene transfer.

Exercise performance improved in all five patients with rest pain orminor ischemic ulcers, who underwent a graded treadmill exercise. Allpatients experienced a significant increase in pain-free walking time(2.5±1.1 min pre gene therapy vs 3.8±1.5 min at an average of 13 weekspost gene therapy, p=0.043) and absolute, claudication-limited walkingtime (4.2±2.1 min vs 6.7±2.9 min, p=0.018). Two patients reached thetarget endpoint of ten minutes of exercise.

Angiography. Digital subtraction angiography showed newly visiblecollateral vessels at the knee, calf and ankle levels in six of tenischemic limbs treated. The luminal diameter of the newly visiblevessels ranged from 200 μm to >800 μm, although most were closer to 200μm and these frequently appeared as a “blush” of innumerablecollaterals. Collaterals did not regress in follow-up angiograms.Magnetic resonance angiography showed qualitative evidence of improveddistal flow with enhancement of signal intensity as well as an increasein the number of newly visible collaterals in eight limbs.

Change in limb status and ischemic rest pain. Therapeutic benefit wasdemonstration by regression of rest pain and/or improved limb integrity.The frequency of ischemic rest pain expressed as afflicted nights perweek decreased significantly (5.9±2.1 at baseline vs 1.5±2.8 at eightweek follow-up, p=0.043), with a slight reduction of analgesicmedication (on average from 1.8 to 1.5 analgetics/24 h period). Based oncriteria proposed by Rutherford, limb status improved in nine of tenextremities treated. Moderate improvement, including both an upwardshift in the clinical category (at least one clinical category inpatients with rest pain and at least two categories to reach the levelof claudication in patients with tissue loss) and an increase in theABI>0.1 was documented in five cases. In one patient an ischemic ulcerresolved sufficiently to permit placement of a split-thickness skingrafting, leading to absolute limb salvage. In two patients, in whom amajor amputation would have been inevitable, retention of a functionalfoot by a minor (toe) amputation was reached. Minimal improvement,including an upward shift in clinical category or improvement of theABI>0.1 was present in another three cases. However, in two patientswith an extensive forefoot necrosis and osteomyelitis a below-kneeamputation was required despite significant hemodynamic and angiographicimprovement. There was one patient with progressive toe gangrene, whoremained unchanged from his hemodynamic and angiographic findings. Thepatient underwent a below-knee amputation eight weeks after genetherapy.

Immunohistochemistry and molecular analysis. Tissue specimens derivedfrom one amputee ten weeks after gene therapy showed foci ofproliferating endothelial cells. This finding was particularly strikingwith the fact in mind, that endothelial cell proliferation is nearlyabsent in normal arteries, is consistent with an estimated endotheliascell turnover time of “thousands of days” is quiescent microvasculature.PCR performed on these samples indicated persistence and widespreaddistribution of DNA fragments unique to phVEGF₁₆₅. Noteworthyamplification of DNA fragments was shown in muscle and skin samplesderived from the site of injection as well as in several muscle samplesremote from the site of injection. Southern blot analysis confirmedpersistence of intact plasmid DNA in muscle specimen derived from twoamputees eight and ten weeks after gene therapy.

Experimental Design

A. Objectives

The scope of this double blind phase III study is to compare thetreatment of intramuscular (IM) gene transfer of phVEGF_(165s) inpatients with critical limb ischemia with the standard treatmentobservation.

1. Primary Objectives

To determine the effect of intramuscular administration of 4000 μgphVEGF₁₆₅ on clinical, physiological parameters of critical limbischemia and safety compared to the standard treatment.

2. Secondary Objectives

To determine and compare the effect on the quality of life for bothtreatments.

B. Selection of Patients

1. Inclusion Criteria

A total of 60 adult diabetic men and women will participate in thisstudy. The restriction for diabetic patients provides a homogeneicpopulation with similar vascular problems (peripheral vessels,frequently no alternative treatment, enough patients to participate).Subjects will be eligible if they have critical limb ischemia and havebeen judged not to be optimal candidates for surgical or percutaneousrevascularization. Subjects must meet the following criteria to beeligible for study enrollment:

Male or female>18 years of age.

Both type I and type II diabetes mellitus patients are candidates.

Presence of critical limb ischemia, according to the followingdefinitions (European Consensus Group on Critical Limb Ischemia):

-   -   rest pain and/or ischemic skin lesions, lasting for more than 2        weeks.    -   ankle systolic blood pressure <50 mmHg, or, in case of        incompressible ankle vessels, toe systolic blood pressure <30        mmHg (44).

Not optimal candidates for surgical or percutaneous revascularization asdetermined by angiography.

2. Exclusion Criteria

Subjects who meet any of the following criteria will be excluded fromthe study enrollment:

Acute surgery necessary.

Pregnancy, lactation, or use of inadequate contraception.

Evidence of cancer (except low grade and fully resolved non-melanomaskin malignancy)

Preproliferative or proliferative diabetic retinopathy, or conditionsobscuring ophthalmological inspection of the retina (e.c. cataract),based on fluorescein angiography of the retina. Patients withoutretinopathy or with background retinopathy are allowed.

Serum creatinine>200 μM.

Concurrent participation in a study using an experimental drug or anexperimental procedure within the 28 days before VEGF gene transfer.

Other severe concurrent illness (e.g., active infection, severecongestive heart failure, or left ventricular ejection fraction[EF]<20%).

Bleeding diathesis, HIV infection, or any other condition that, in theopinion of the investigator, could pose a significant hazard to thesubject if the investigational therapy was to be initiated.

Inability to follow the protocol and comply with follow-up requirements.

C. Dose and Administration

1. Dosage

The clinical study is performed as a double blind phase III study in 60patients. In half of the patients a total dose of 4000 ug ph VEGF₁₆₅will be given, each patient will receive 4 injections (each with 500 μgphVEGF₁₆₅), each with a volume of 1 ml. This will be repeated after 4weeks (day 28). The other half of the patients will receive injectionswith physiologic salt without phVEGF.

2. Administration

The VEGF gene via a small needle (27 G) will be injected directly intothe muscle. With echography the position of the needle will be checked.The spreading of the injectate into the muscle will also be monitored inthis way. After introduction of the needle 5 ml of saline or salinecontaining 500 μg phVEFG₁₆₅ will be injected. This will be done at fourdifferent injection sites in gastrocnemius and anticus muscles.

D. Concomitant Therapy

No concomitant routine therapy will be excluded in this study. Subjectswill be treated by their personal physicians with routine medication asneeded. No restriction of medication will be stipulated. However,physicians will be encouraged not to change the established regimen ofanalgetics post intramuscular injections unless clearly indicated by achange in clinical status.

E. Pretreatment Assessments

Subjects will be screened prior to study initiation to determine theireligibility for the study based on the inclusion and exclusion criteria.Written informed consent will be obtained from subjects prior to intramuscular injections. To be eligible, the following assessments must beperformed/obtained within 2 weeks prior to intramuscular injectionunless otherwise indicated:

Medical history, including medications, demographic information,vascular history, and history of cancer.

Review of systems and specific emphasis on symptoms or historysuggestive of tumors and on reproductive status.

Pain assessment by the McGill Pain Questionnaire and the pain ratingindex (PRI).

Vital signs (blood pressure, pulse, respirations, temperature).

Physical Examination:

-   -   Vascular status, using angiography and noninvasive vascular        laboratory examinations, including ankle and toe pressures and        Doppler spectral analysis or duplex examination of large lower        extremity vessels. Criteria for CLI see above.    -   Detailed ophthalmological examination, including funduscopy,        fundus photographs, and fluorescein angiography of retinal        vessels.    -   Neurological examination, including quantified muscle force        testing, neurographic examination of motor and sensory nerves of        arm and leg, and quantified sensory examination.

Clinical Laboratory Samples:

-   -   SMAC-20 (sodium, potassium, bicarbonate, chloride, glucose, BUN,        creatinine, albumin, alkaline phosphatase, bilirubin, calcium,        creatine kinase, LDH, phosphate, SGOT [ALT] SGPT [AST],        tropinins, CRP, total protein, uric acid, cholesterol,        triglycerides; VEGF protein and plasmid; anti-dsDNA, two nightly        portions of urine for micro-albuminuria).    -   Additional laboratory assessments:        endothelial>markers=including: factor VIII-vWF, ICAM, VCAM,        e-selectin.    -   CBC with manual differential and platelets.    -   Complete urinalysis, including dipstick for protein.

PT/aPTT or INR.

12-lead ECG.

Chest X-ray (posterior-anterior and lateral).

Serum pregnancy test for women of childbearing potential (within 24hours prior to study drug administration).

F. Follow-Up Assessments

In hospital till three days after the injections: daily, vital signs,physical examination, use of analgetics, electrocardiogram, clinicallaboratory testing (SMAC 20).

Follow-up clinic or office visit will be conducted when possible on anoutpatient basis on Days 3, 7, 14 and 72 days after the intra muscularinjections. The following assessments will be performed/obtained on Days3, 7, 14 and 72 days unless otherwise indicated:

Vital signs (blood pressure, pulse, respirations, temperature).

Physical examination, including funduscopy.

Interval history (including medication changes) and review of systems.

Record the analgetics use in a diary.

Clinical laboratory samples SMAC-20 CBC with manual differential andplatelets, urinalysis.

F1. Schedule of Evaluation

1×/4 mo Day until 2 Day 0 28 yr Pretreatment VEGF Day VEGF Day Day DayDay after 1^(st) Day 0 im Day 3 Day 7 14 im 31 35 42 100 injectionHistory X X X X X X X X X X (analuse) QLC and X X X X X pain Vital X X XX X X X X X signs Physical X X X X X X X X X X examination ‘SMAC- X X XX X X X X X 20’ Vascular X X X X X X X techniques Angiography X Xophthalmological X X X examination Neurologic X X examination PET scan XXG. Analysis of Clinical Response

Patients will receive a diary to note their severity of pain (painassessment by the McGill Pain Questionnaire and the PRI) and the dailyuse of analgetics. In the diary they also note their daily medicationand activity.

H. Analysis of the Quality of Life

In this study the RAND-36 will be used to analyze the quality of life.

I. Analysis of Physiological and Anatomical Activity

On the specific vascular methods, and the choice of primary andsecondary measures of effect:

The clinical course of an episode of CLI may be quite variable. Althoughin some patients an inexorable downhill course to amputation follows, inothers slow healing of ulcers may occur. Several risk factors for anadverse outcome have been identified, such as diabetes, continuedsmoking, concurrent other cardiovascular disease or vascular laboratoryparameters like low ankle or toe pressure or TcPO₂. However,unpredictability of ulcer healing in an individual patient stresses theimportance of a controlled design in an intervention study. This is alsothe major flaw in the clinical studies of VEGF gene transfer in CLIreported so far.

The increase in ankle pressures and ABI reported in the studies of Isneret al of VEGF gene therapy in in CLI is remarkable. In factnon-revascularization interventions in CLI (medication, training) havenever been reported to result in consistent increases in anklepressures. In revascularisation studies a 15% increase in anklepressures is considered to be clinically relevant (45, 46).

I1. Available Vascular Techniques.

For the primary measure of effect:

I1.1. Ankle- and Thigh-brachial and Toe-brachial Pressure and Index.

Using standard techniques and following AHA recommendations, the Dopplerankle blood pressure is measured and divided by a simultaneouslymeasured brachial artery blood pressure (47). In short, using a 8 MHzDoppler probe forming part of a Parkes Doppler equipment audio Dopplersignals are obtained over the posterior tibial and dorsalis pedisarteries of both feet. After suprasystolic inflation of previouslyapplied cuffs adapted for ankle (12 cm wide) and thigh (18 cm wide)circumference, the ankle (and optionally: thigh) Doppler systolic bloodpressure are measured during slow deflation. Measurements are made atrest. If the foot condition allows so, the measurement at rest will befollowed by a measurement immediately after 3 minutes of exercise on acalf ergometer with a moment of 33 Nm. The toe pressure is determinedusing a toe cuff with photoplethysmographic assessment of the systolicblood pressure level during cuff deflation. Using an automated bloodpressure meter simultaneous brachial artery blood pressures areobtained. The ankle-, thigh- and toe-brachial index is calculated bydividing the ankle blood pressure by the simultaneously measuredbrachial artery blood pressure. For the ankle blood pressure the highestof either posterior tibial or dorsalis pedis artery at rest is used, forthe measurement after exercise the same foot artery is used. The CV ofthe measurements at rest has been found in several studies to be around5% for intra-individual day-to-day comparisons (48,49,50). Forinter-individual comparisons of technicians the CV amounts to 8% (48).For a post-exercise ankle-brachial index the CV amounts to 9% (51).

For secondary measures of effect:

I.1.2. Flow in Calf/Forearm Using Strain Gauge Plethysmography.

Calf blood flow. Resting and maximal calf blood flow can be measurednon-invasively by plethysmography, using an ECG-triggered venousocclusion plethysmograph with a pneumatically powered cuff inflatorallowing rapid flow measurements. Flow measurements at rest are made insubjects by alternate occlusion and deflation of the cuffs duringintervals of 5–6 heart beats, maximal blood flow is determined duringthe hyperemic response immediately following exercise. Measurements ofthe flow during exercise cannot be performed simply with venousocclusion plethysmography because the muscles move strongly and thevolume changes during the contractions. Measurements of post-exercisehyperaemia, on the other hand, can be performed technically simply. Calfor forearm blood flow can be calculated from the rate of the initialincrease in calf or arm circumference during venous occlusion and isexpressed as milliliters per 100 ml of calf tissue per minute (52, 53,54). Equipment is available for bilateral measurements at the Vascularlaboratory of the Department of Medicine.

I.1.3. Skin Flow Assessment Using Laser Doppler Flowmetry.

Laser Doppler flowmetry is a technique for measuring changes in tissueblood flow. The method is based on the Doppler shift of monochromaticlaser light scattered back by tissue. The Doppler signal is determinedby the velocity and the number of moving scattering particles, mainlyred blood cells and therefore is directly related to tissue blood flowrate. This non-invasive method is applicable to almost every tissue,although it has until now mainly been used for measuring skin bloodflow. In the present study we will use a Diodop LDF meter, connected toa PC with self-developed software for signal analysis. Measurements willbe made at rest after 3 minute of suprasystolic occlusion. A biologicalzero will be obtained. Local skin temperature will also be recordedusing an Ellab thermocouple thermometer. Measurements will be performedat the medial malleolus of the treated leg, in case open lesions arepresent another site at the foot will be used (55, 56, 57, 58).

Optional (if possible combined with fluorescein angiography of theeyes):

I.1.4. Fluoresceine Capillary Microscopy.

In the skin the transcapillary diffusion of intravenously injectedfluorescein is used to measure vascular permeability. Using singlecapillary fluorescein videodensitometry, Bollinger et al. havedemonstrated that fluorescein diffusion in the skin is increased in agroup of subjects with long-term diabetes mellitus (Boilinger 1982).This observation was confirmed by the same group, using a “large window”method (which is comparable to the method we used in our study), inwhich a larger number of capillaries, up to approximately 100, isstudied. The large window method is, therefore, less sensitive forspatial and temporal changes in capillary flow. We have considerablyimproved the reproducibility of this method and thereby its value as atool in intervention studies, without sacrificing its power todiscriminate diabetic from normal subjects. Use is made of custom-madeequipment, described elsewhere in detail (59, 60, 61, 62).

I.1.5. Positron Emission Tomography (PET).

Local perfusion and reduction of ischemic areas are to be detected byPET; H₂O¹⁵-PET perfusion) or F-deoxyglucose (metabolism).

I.2. Ophthalmic Examination.

The following ophthalmic examinations will be conducted and recorded:Ophthalmic history, best corrected visual activity, slit-lampbiomicroscopy, intraocular pressure, fundus examination,fundusphotography and fluorescein angiography. Patients are categorizedaccording to their clinical examination in having no retinopathy,background, preproliferative and proliferative diabetic retinopathy,based on the ETDRS international classification. Seven standard fundusphotographs of both eyes are made through dilated pupils.

Fluorescein angiography in performed of the macular area of the righteye and of the mid periphery of both eyes in the late phases (64, 65,66).

I.3. Neurologic Examination.

Neurological examination, the same measurements as performed atpretreatment assessment.

J. Follow-up Assessments:

-   -   Physical examinations.    -   Vascular techniques: angiography, ankle-, (optional) thigh-, and        toe pressures and -brachial index, laser Doppler flowmetry.        Before and at the end of follow-up.    -   Ophthalmological examination; funduscopy and fundus photographs.    -   Neurological examination.    -   SMAC-20.        K. Potential Side Effects.

A. Complications related to diagnostic angiography such as rupture of anartery, infection, embolization, or allergic reactions to the contrastmedia used.

B. Even though we have attempted to minimize that risks associated withgene transfer by eliminating the need for any viral, liposomal or othervectors, it is recognized that theoretical risks of gene transferremain. Though the DNA to be transferred is considered to be harmless,events could occur within normal cells that take up the foreign DNA thatallow them to be transformed. Laboratory studies suggest that this isunlikely. We nevertheless acknowledge that cells could theoreticallybecome abnormal after long periods of time.

C. Complications related to the VEGF protein. Until now only oedema ofthe treated limb has been described as minor complication. No evidencefor systemic complications such as induction/exacerbation.

L. Definition of Response

Response will be defined as limb survival and/or improvement in ABI of15%, as two independent variables. Limb survival is defined as absenceof major amputation. Major amputations are those at the level of theankle of higher.

M. Risk-Benefit Analysis

In the patient the major benefit we anticipate is to prevent majoramputation. The risk can be toxicity as nephropathy, retinopathy andoedema and secondary infections due to the local injections.

On the basis of pre-clinical animal studies, we do not anticipateadverse consequences.

In the absence of gene transfer, deleterious consequences might includepersistent rest pain, deterioration of the limb ischemia.

N. Evaluation of Safety

Following intramuscular injection toxicity will be evaluated to WHOcriteria. No further injections will be given in case of any grade 3 or4 toxicity with is caused by the intramuscular injections.

O. Statistics

The natural history for the need for major amputations of nonreconstructable CLI (with ankle systolic pressure <50 mm Hg) isapproximately 50% in 100 days. Based on the results of Isnerdemonstrating an approximately 50% salvage rate in limbs destined to beamputated when VEGF started, we hope for a substantial improvement inamputation rate. As treatments starts early we estimate that at least50% of limbs at risk will not be deteriorate to the point of majoramputation. From these patients who deteriorate a 50% salvage rate mightstill be feasible. Therefore, an improvement of approximately 50% majoramputation to 12.5% is expected in this study. To detect the expected37.5% improvement of limb survival (50% amputations to 12.5%) or a 50%improvement of ABI of 15% with the treatment of VEGF compared to thecontrol group, 26 patients are needed in both groups (significance levelof 0.05 (one-sided) and power 0.85; according to a binomialdistribution). In total 60 patients will be entered because of a dropout percentage of appr. 10% in these group of patients. There will be ablock randomization for two times 30 patients. After treating 30patients there will be an interim analysis. The study will be stopped ifthere is a significant difference between both groups after 30 patientshave been entered.

P. Subject Discontinuation

Subjects may discontinue the study at any time. If, in the judgment ofthe investigators, continuation in the study would be detrimental to thesubject, the investigator may withdraw the subject at any time.

If a subject withdraws from the study after 24 hours, but before thesafety and biologic activity follow-up period, he or she should becontacted in order to obtain information about the reason(s) for his orher withdrawal and the occurrence of any adverse events. The subjectshould be urged to return to the clinic for an early discontinuationvisit to be assessed for safety and biologic response.

Q. Randomisation

This is a double blind placebo controlled phase III study. The placebois produced in the pharmacy. There will be a block randomization for twotimes 30 patients and an interim analysis. Randomization will be done bysealed envelope method without replacement.

R. Results:

Preliminary results show that administration of VEGF gene to patientswith critical limb ischemia has a distinct positive effect as comparedtot the control group.

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1. A vector comprising a DNA sequence encoding human vascularendothelial growth factor, wherein said nucleic acid sequence comprisesnucleotides 682–1258 of SEQ ID NO: 6 operably linked to a modified pCMVpromoter of nucleotides 14–619 of SEQ ID NO:
 6. 2. An isolated host cellwhich comprises a vector according to claim
 1. 3. An isolated host cellaccording to claim 2, which is a human cell.
 4. A composition comprisinga vector according to claim
 1. 5. A pharmaceutical compositioncomprising a vector according to claim 1 and a pharmaceuticallyacceptable carrier.
 6. A method of treatment of arterial diseases,comprising intramuscularly administering a vector according to claim 1with a carrier to a suitable recipient, wherein production of said VEGFresults in an increase in the number of collateral blood vessels inischemic tissues and/or improved blood flow.
 7. A method of treatment ofarterial diseases, comprising intramuscularly administering acomposition according to claim 4 or 5 to a suitable recipient, whereinproduction of said VEGF results in an increase in the number ofcollateral blood vessels in ischemic tissues and/or improved blood flow.